The One-carbon Carrier Methylofuran from Methylobacterium extorquens AM1 Contains a Large Number of α- and γ-Linked Glutamic Acid Residues

Abstract

Methylobacterium extorquens AM1 uses dedicated cofactors for one-carbon unit conversion. Based on the sequence identities of enzymes and activity determinations, a methanofuran analog was proposed to be involved in formaldehyde oxidation in Alphaproteobacteria. Here, we report the structure of the cofactor, which we termed methylofuran. Using an in vitro enzyme assay and LC-MS, methylofuran was identified in cell extracts and further purified. From the exact mass and MS-MS fragmentation pattern, the structure of the cofactor was determined to consist of a polyglutamic acid side chain linked to a core structure similar to the one present in archaeal methanofuran variants. NMR analyses showed that the core structure contains a furan ring. However, instead of the tyramine moiety that is present in methanofuran cofactors, a tyrosine residue is present in methylofuran, which was further confirmed by MS through the incorporation of a 13 C-labeled precursor. Methylofuran was present as a mixture of different species with varying numbers of glutamic acid residues in the side chain ranging from 12 to 24. Notably, the glutamic acid residues were not solely ␥-linked, as is the case for all known methanofurans, but were identified by NMR as a mixture of ␣-and ␥-linked amino acids. Considering the unusual peptide chain, the elucidation of the structure presented here sets the basis for further research on this cofactor, which is probably the largest cofactor known so far.

Lanthanide-Dependent Regulation of Methylotrophy in Methylobacterium aquaticum Strain 22A

“…Methylene-H 4 MPT can be formed spontaneously or by the formaldehyde-activating enzyme, which is further transformed to formate via the H 4 MPT pathway. Methylofuran was recently identified in strain AM1, and it carries a C 1 unit from formyl-H 4 MPT in the pathway ( 26 ). The genes involved in the pathway were all upregulated under the MCa condition compared to the SCa condition, but were relatively repressed in the presence of La 3+ compared to the MCa condition.…”

Methylofuran is a prosthetic group of the formyltransferase/hydrolase complex and shuttles one-carbon units between two active sites

“…Both, MFR and MYFR contain a furan moiety with a formylatable aminomethyl group. However, compared to the five known archaeal MFR derivatives (10–13), the core structure of MYFR contains a tyrosine residue instead of the tyramine and has a polyglutamic acid side chain consisting of between 12 and 24 glutamates attached (5), significantly more than what has been described for MFRs (up to 12 γ-linked glutamates in the case of MFR-d) (13). In addition, the glutamic acid residues of MYFR were identified as a mixture of α-linked and γ-linked amino acids.…”

“…The metabolism of methanogenic, methanotrophic, and sulfate-reducing archaea and methylotrophic bacteria hinges on coenzymes that bind, convert, and release one-carbon units (1–4). Two of these coenzymes, methanofuran (MFR) and tetrahydromethanopterin (H 4 MPT), are present in both the domains of Archaea and Bacteria (3, 5). Oxidation levels of coenzyme-bound one-carbon intermediates are shared in anaerobic methanogenesis and aerobic methylotrophy (Fig.…”

“…The analog of MFR present in the methylotrophic bacterium Methylorubrum extorquens (formerly Methylobacterium extorquens ; ref. 9) has recently been identified and termed methylofuran (MYFR) (5). Both, MFR and MYFR contain a furan moiety with a formylatable aminomethyl group.…”

Wide Distribution of Genes for Tetrahydromethanopterin/Methanofuran-Linked C1 Transfer Reactions Argues for Their Presence in the Common Ancestor of Bacteria and Archaea

“…This gene is also not recognizable in Methylobacterium genomes. As methanofuran can be measured in these bacteria (Hemmann et al, 2016 ), a non-homologous substitution must be present.…”